PREPARATION AND MATERIALS
PER GROUP OF FOUR STUDENTS:two sea urchins, syringe, 2 small beakers (50 ml),
filtered sea water, ph 5,7,8 sea water, CA++Mg++free sea water, hand
centrifuge, container of ice.
THIS INVESTIGATION REQUIRES: SLIDES and coverslips
ANIMALS:sea urchins (Echinodermata)
UTENSILS:50 ml beakers, syringe and needle for injection of sea urchins,
centrifuge tubes, pipettes, petri plates.
PRELAB; LOOK UP integrin, BINDIN, VITELLINE MEMBRANE, SPERM
MOTILITY, ACROSOME REACTION, CORTICAL REACTION.
OBJECTIVES: to see sperm
movement, attachment to egg, lifting away of fertilization membrane from
surface of egg, first cleavage and to investigate mechanisms involved.
REQUIRED TASKS: fertilize
eggs, observe fertilization membrane lifting off the surface of egg.
OBSERVE EFFECTS OF PH,
TEMPERATURE, CA++ CONTENT ON FERTILIZATION. TEST THE EFFECTS OF ‑SH
AGENTS ON FERTILIZATION MEMBRANE HARDENING. Students to work in pairs and turn
in completed lab sheet at the end of the lab
PROCEDURES: In this
section you will determine the relationship between eggs and sperm and correct
environmental conditions required for normal fertilization, fertilization
membrane formation, and first cleavage
LABEL ALL DISHES OF EGG FERTILIZED WITH THE TIME OF SPERM ADDITION, AND
ANY VARIABLE CONDITION. MAKE SURE YOU HAVE AN UNTREATED CONTROL WHICH HAS NO
SPERM ADDED, IF THAT IS REQUIRED IN YOUR EXPERIMENT.
EACH GROUP OF 2 STUDENTS;
1)
1.inject
two sea urchins with 1 cc of .55 M KCl and then give them a good shake and wait
to see what sex they are: the gametes come out five gonopores at the aboral
surface (opposite the oral surface where the mouth is) if the gametes are
white, it is sperm and should be shed by turning the male upside down in a
petri dish placed on ice; if the gametes are yellow, it is a female and the eggs
should be shed into filtered sea water (pH 8) by inverting them over a beaker
full of sea water. The beaker should be of smaller diameter than the sea urchin
so that it won't fall in. Keep injecting until you get both sexes, after
appropriate waits to see what the next one is. You can trade with other groups,
also.
ALWAYS KEEP THE EGGS AT ROOM TEMPERATURE AND THE SPERM ON ICE
This means use room temperature sea water in all egg treatments and at
fertilization. If it is cold, warm it by setting it in a pan of warm
water. Give another 1 cc injection when
egg spawning slows down, then leave the female to complete the spawning for
about ten more minutes. In the meantime, design your experiments and label your
dishes.
FACTORS
INVOLVED IN FERTILIZATION
The
release of calcium from cytoplasmic stores and the entry of calcium from the
sea water increase the content of calcium in both sperm and eggs which signals
them to secrete. In the case of the sperm, it is the acrosome reaction. In the
case of eggs it is the cortical reaction of cortical granules. The changes are
in response to sperm-egg interaction at receptors on the surface of the sperm
and eggs which produce second messengers, and in response to electrical changes
in membrane potential which open voltage regulated ion channels. To test for
which ones are working at fertilization, we can add drugs to the sperm or the
eggs to pretreat them before they interact.
CALCIUM
CHANNEL DRUGS: these drugs lead to polyspermy since they prevent lifting of
fertilization membrane.
references-
McCulloh,
DH, Ivonnet, PI, Landowne, D, Chambers, EL 2000. Calcium entry mediates the
voltage dependence of sperm entry into sea urchin eggs Devel. Biol 223:449-62.
They used
2
ul dry sperm per 40 ml SW
DRUGS WHICH PREVENT CORTICAL REACTION:
All
drugs were dissolved in DMSO at concentrations
that when diluted with SW, DMSO not more than .125%
Organic.
They all promote sperm entry when eggs clamped at neg voltage- they relieve the
block to sperm entry by blocking Ca entry and therefore the cortical
reaction of eggs.
Diltiazem-benzothiazipine 50 uM
Nifedipine-dihydropyridine 30 uM
Nimodipine
–dihydropyridine 50 uM
Inorganic
blockers- made up in sulfate-free SW also promote sperm entry on neg clamped
eggs
Cd++
100 uM
Ni++
Blocker of Ca release from
endoplasmic reticulum:
want to check for ruthenium
red block of cortical reaction and acrosome reaction by sperm due to block of
IP3 receptor which is a calcium channel in ER.
CALCIUM IONOPHORE a23187 is
a calcium channel open in membrane
which allows the acrosomal reaction without contact with egg jelly and cortical reaction without sperm-
artificial activation.
Platelet Activating factor
(PAF) also causes the acrosomal reaction and cortical reaction without sperm
through its receptor and second messenger system.
The following is a layout of
an experiment to study the effects of one of the drugs:
|
|
Incubate
sperm with drug or DMSO for 2min before add other |
Preinc
for 2 min |
|
|
|
Add
after 2 min drug |
|
|
Add
after 2 min drug |
|
tube |
Sperm
alone |
Ruthenium
red- want 50 uM final use o.5 mM in sea water |
eggs |
Drug Final
50 uM |
DMSO
.2% |
PAF
2 mM- 100 uM final |
sperm |
SW |
Jelly
from eggs |
|
1 |
SW |
|
|
|
|
|
50
ul |
.5 |
|
|
2 |
drug
nojell |
|
|
2ul |
|
|
50
ul |
.5 |
|
|
3 |
DMSO
no |
|
|
|
2
ul |
|
50
ul |
.5 |
|
|
4 |
jelly |
|
|
|
|
|
50
ul |
|
.5 |
|
5 |
Jell,drug |
|
|
2ul |
|
|
50
ul |
|
.5 |
|
6 |
jelldmso |
|
|
|
2ul |
|
50
ul |
|
.5 |
|
7 |
jellruth |
50
ul |
|
|
|
|
50
ul |
|
.5 |
|
8 |
PAF |
|
|
|
|
5ul |
50
ul |
.5 |
|
|
9 |
DRUG,PAF |
|
|
2ul |
|
5ul |
50
ul |
.5 |
|
|
10 |
DMSO,PAF |
|
|
|
2
ul |
5ul |
50
ul |
.5 |
|
|
11 |
Ruth
paf |
50
ul |
|
|
|
5
ul |
50
ul |
.5 |
|
|
|
Eggs
plus sperm |
Ruthenium
red- want 5 uM final use o.5 mM |
eggs |
DRUG Final
50uM Use
5 mM |
DMSO
.2% |
PAF
2 mM- 10 uM final |
sperm |
SW |
jelly |
|
|
SW |
|
.5 |
|
|
|
50
ul |
|
|
|
|
DRUG |
|
.5 |
2ul |
|
|
50
ul |
|
|
|
|
DMSO |
|
.5 |
|
2
ul |
|
50
ul |
|
|
|
|
RUTHRED |
50
ul |
.5 |
|
|
|
50
ul |
|
|
|
|
Eggs alone
|
Ruthenium
red- want 50 uM final use o.5 mM |
eggs |
DRUG Final 50uM use
5 mM |
DMSO
.2% |
PAF
2 mM- 10 uM final |
sperm |
SW |
jelly |
|
|
Paf
ruth |
50
ul |
.5 |
|
|
5
ul |
|
|
|
|
|
PAF |
|
.5 |
|
|
5ul |
|
|
|
|
|
DRUG,PAF |
|
.5 |
2ul |
|
5ul |
|
|
|
|
|
DMSO,PAF |
|
.5 |
|
2
ul |
5ul |
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
2)
After spawning is complete pour off the sea
water into another beaker, so you don't lose your eggs by mistake. Pour the
remainder into a centrifuge tube and spin 15 rotations on the hand centrifuge
(make sure you have a balance tube opposite.) REMOVAL OF EGGS FROM THE
CENTRIFUGE TUBES CAN DAMAGE THEM UNLESS IT IS DONE CORRECTLY! Always pour some
fluid over the eggs, then using a pasteur pipette with a rubber bulb, fill the
pipette with fluid from the top of the tube, then gently force the fluid out
into the bottom of the tube to stir the eggs. Do not let air bubbles do the
stirring, and don't create air bubbles or suck the eggs up into the pipette and
then back out‑ it homogenizes them.
Removal of jelly: Add 25 ml PH5
sea water to empty the eggs from the tube into the beaker, and allow it to stand
for three minutes before adding .5 ml 1 M tris pH 8 to raise the pH rapidly.
(the pH 5 treatment removes the egg jelly, but prolonged treatment leads to
damage to the eggs.) Now we must remove the dissolved egg jelly by washing the
eggs. So do centrifugations as above, SAVING THE FIRST SUPERNATE, LABEL IT
JELLY, and wash the eggs with a tube full of sea water pH 8, 2 times,
suspending the eggs each time as above.
3) Now we have our eggs prepared for the experiment. Sperm are much more delicate than the eggs, so they must be kept undiluted in ice (the seminal fluid has substances in it to keep the sperm inactive and the ice also helps to do that.) They must be diluted prior to their addition to the eggs, or we get polyspermy and abnormal cleavage. Add one drop of concentrated sperm to 10 cc of sea water in a graduated cylinder, stir to disperse evenly. This diluted sperm suspension will only last for 20 minutes, so do it right before you need it, and keep it in the ice bath. Prepare a slide with a drop of eggs, have it set up on a microscope prior to seprm addition adjusting the light the eggs on the slide easily visible. Add a drop of the sperm, slam the cover slip on the slide and place under the low power objective and observe fertilization. If you don't get fertilization try it again with another batch of sperm from someone who found good fertilization. Then proceed to fertilize your aliquots of eggs.
4) Do as many of the experiments as you can, but ask some other groups to do drugs you didn’t and then look at their results too.
5)
Fertilization:
ADD DILUTED SPERM TO mini-tubes as in table above with SEA WATER and EGGS.
Observe, using dissection microscope and dark field (obtained by putting mirror
on its side until field appears black and eggs appear white) or compound microscope. The fertilization
membrane is formed by a lifting away of the vitelline membrane, and an addition
of material to it from the ruptured and released contents of the cortical
granules. Look at the number of sperm
around one egg, record for a few eggs. Observe and record the sperm shape and
size compared to the egg, movement of the flagellum. For polyspermy, use a dish of eggs
with one of the calcium channel drugs. Check the effect on cleavage, as
compared to the control.
6)
Test the effect of pH on fertilization.
Fertilize a DISH of eggs in the same manner, but use pH 5 sea water in one
dish, pH 7 in another. Record the % fertilization membranes formed after 1 min,
5 min. What is your control?
IF YOU ARE GOING TO CULTURE THE EMBRYOS
TO LATER STAGES, DECANT OFF THE SPERM LADEN SEA WATER ATER THEY SETTLE, AND
POUR A SHALLOW LAYER INTO PYREX BAKING DISHES AND INCUBATE THEM AT 15 DEGREES C
FOR ANYTHING PAST THE 2 CELL STAGE OR THE CLEAVAGES WILL NOT BE SYNCHRONOUS. IT
TAKES ABOUT 1 1/2 HRS TO GET TO THE FIRST CLEAVAGE. KEEP CHECKING TO SEE IF YOU
CAN SEE THE MITOTIC APPARATUS DEVELOPING. The fertilization membrane is formed
by a lifting away of the vitelline membrane, and an addition of material to it
from the ruptured and released contents of the cortical granules. Look at the number of sperm around one egg,
record for a few eggs. Observe and record the sperm shape and size compared to
the egg, movement of the flagellum. What is your control? Why is the lifting of
the fertilization membrane a good indication that a sperm has penetrated the
egg?
If you are going to culture the embryos
to later stages, decant off the sperm laden sea water after they settle, and
pour a shallow layer into pyrex baking dishes and incubate them at 15 degrees c
for anything past the 2 cell stage or the cleavages will not be synchronous.
It takes about 1 1/2 hrs to get to the
first cleavage. Keep checking to see if you can see the mitotic apparatus
developing.
Keep in mind that we have two sets of
factors working in fertilization: 1) the activation of the sperm acrosome
reaction, polymerization of actin filament 2) activation of egg and cortical
reaction. Try to figure out which require Ca++ release and which require a pH
change. Calcium ionophore and NH4 can be used to answer these questions.
reference-Nucciteli:
How do sperm activate eggs? Current topics in developmental biology vol 25.
Chapter 1. 1991.
1)
EFFECT OF TEMPERATURE ON FERTILIZATION. Fertilize a dish of eggs
that has been sitting on ice for ten minutes. Compare the percent fertilization
after and 5 min. with the control. .UL
EFFECT OF CA++ ON FERTILIZATION. Fertilize a dish of eggs using Ca++Mg++free
sea water instead of regular sea water. Compare the results with the control.
2)
EFFECT OF ‑SH REAGENTS ON VITELLINE MEMBRANE AND FERTILIZATION
MEMBRANE.
treat the eggs with DTT or glutathione prior to fertilization, and check them
after addition of sperm.
3)
METHODS FOR REMOVING VITELLINE MEMBRANE:
1. Epel, D., AM Weaver, D. Mazia. 1970. Exp cell res 61:64-68. Methods for removal of the vitelline
membrane of sea urchin eggs. I. Use of Dithiothreitol (Cleland Reagent.)
Incubate unfertilized S. purp or L. Pictus or Dendraster excentricus in
5 mM DTT at pH 8.0. Releases jelly, and vitelline m and accessory cells of
starfish. 5 min to remove jelly and VM. pH very important. higher pH (9.2)
requires 1/2 the time.
2. BM Shapiro. 1981. Awakening of the invertebrate egg at
fertilization. In Fertilization and embryonic development in vitro. Eds.
L Mastroianni, Jr. and JD Biggers. Plenum Press, NY. pp233-255.
used 3 amino-1,2,4-triazole to inhibit ovoperoxidase activity, FM
remains soft and can be removed anytime (Showman and Foerder, 1979:Exp Cell
Res. 120:253-255. Removal of the fertilization membrane of sea urchin embryos
employing aminotriazole.)
3. SG Ernst, BR Hough-Evans, RJ Britten, and EH Davidson. 1980. Limited
complexity of the RNA in micromeres of sixteen-cell sea urchin embryos. Devel.
Biol. 79:119-127.
fert at 5x10 E5 eggs/ml add equal vol of following after membrane
elevates (about 90 sec):freshly prepared .08% papain, .40%glutathione, pH 7.8,
swirl gently and look until membranes begin to disappear 90-120 sec later.
After 7-9 min dilute to 1-3x10 E4.
4. RO Hynes and PR Gross. 1970. A method for separating cells from
early sea urchin embryos. Devel. Biol. 21:383-402.
fert eggs in 0.04% papain, .2% cysteine, sea water, pH 7.8. Make fresh
daily. Keep eggs suspended 4-5 min, spin down gently, remove supernate, wash 2x
with filtered SW
What do these
methods tell us about how the vitelline membrane is held on the egg and what happens
at fertilization to lift it off as the fertilization membrane?
PLATELET-ACTIVATING FACTOR CAN ACTIVATE THE CORTICAL REACTION IN SEA URCHIN UNFERTILIZED EGGS AND MEIOSIS IN STARFISH EGGS
Mary
Lee Sparling, Biology Dept Jan, 2001
Platelet-activating
factor (PAF) is a choline phospholipid similar to phosphatidyl-choline, a
membrane lipid, but having an acetyl group on its second carbon instead of a
long acyl hydrocarbon chain. It causes secretion of materials stored in
secretory granules in many kinds of cells (blood platelets, neurons, immune
cells, and follicle cells) and causes the acrosome reaction of sperm. The
effect of PAF is due to a PAF-receptor which when occupied can cause activation
of many enzymes producing lipid second messengers or lipid substrates. PAF is a
very ancient and important lipid signal in reproduction and may aid gamete
union as well as prevent apoptosis of egg and sperm once they are united since
PAF Receptor can activate PLA, PLC, PLD, PI3K, GTPase, Ca++ channel, and PKC. Production of PIP2 by PI3K or activation of
PKC, to phosphorylate many cell proteins, prevents apoptosis. PAF is produced
after fertilization by sea urchin egg homogenates and after stimulation of
meiosis in starfish egg homogenates. It is considered an autocoid, or substance
that when secreted can cause changes to the cell that produced it. Fluorescent
PAF and other related lipids (LPAF, PC, PE) were applied to eggs to see where
they go. The effect of PAF does not seem to require entry into the cell.
Application of 5X10-4 M PAF caused secretion of the cortical
granules which usually only occurs in eggs which have been fertilized. A
similar activation of secretion can be caused by calcium ionophore A23187 which
also activates the enzyme pathway which synthesizes PAF. Normal PAF production
at the time of fertilization may be the cause of the secretion of the acrosome
by sperm and the cortical reaction in normal eggs, and a part of the program for activation of cell division and
differentiation.
Database:
MEDLINE
Author(s): Brandriff B ;
Hinegardner RI ; Steinhardt R
Title: Development and life
cycle of the parthenogenetically activated sea urchin embryo.
Source: J Exp Zool (JOURNAL OF
EXPERIMENTAL ZOOLOGY) 1975
Apr; 192 (1): 13-24
Journal Code: I47
Abstract: A method is reported
for inducing parthenogenetic development in eggs of the sea urchin Lytechinus
pictus, a species which previously could not be artificially activated. NH4OH
or the calcium ionophore A23187 are
used as activating agents followed by
hypertonic treatment. The ionophore is
superior in activating large numbers of
unfertilized eggs, whereas NH4OH produces a larger percent of embryos able to undergo
gastrulation. Both feeding larvae and
urchins arising from these
artificially activated eggs are diploid. All individuals in which sex has been identified have
been female. The viability of these
completely homozygous organisms is low
compared to their
fertilized counterparts.
·
Author(s):
Kamata Y ; Mita M ; Fujiwara A ; Tojo T ; Takano
J ; Ide A ; Yasumasu I
Address: Department of
Biology, School of
Education, Waseda
University, Tokyo, Japan.
Title: Probable participation
of phospholipase A2 reaction in the process of fertilization-induced
activation of sea urchin eggs.
Source: Dev Growth Differ
(DEVELOPMENT GROWTH AND
DIFFERENTIATION) 1997
Aug; 39 (4): 419-28 Journal
Abstract: In sea urchin eggs
activated by sperm, A23187 or melittin, BPB (4-bromophenacyl bromide, a
phospholipase A2 inhibitor) blocked fertilization envelope formation and transient CN(-)-insensitive respiration in a concentration-dependent
manner. BPB had virtually no effect on
the increase in [Ca2+]i (cytosolic Ca2+ level), the activity of phosphorylase a and the rate of protein
synthesis, as well as acid production
and augmentation of CN(-)-sensitive
respiration. BPB also inhibited
fertilization envelope formation and augmentation of CN(-)-insensitive respiration induced by
melittin. Melittin, known to be an activator
of phospholipase A2, induced the
envelope formation,
acid production, augmentation of
CN(-)-insensitive and sensitive
respiration, but did not cause any
increase in [Ca2+]i, the phosphorylase a
activity and the rate of protein
synthesis. An activation of
phospholipase
A2 induced by Ca2+ or melittin seems
to result in cortical vesicle discharge and
production
of fatty acids, which are to be utilized
in CN(-)-insensitive lipid peroxidase reactions.
Activation of other examined cell functions
in eggs activated by sperm or A23187, probably results from Ca(2+)-triggered sequential reactions other
than Ca(2+)-caused activation of
phospholipase A2.
Author(s): Elhai J ; Scandella CJ
Title: Arachidonic acid and
other fatty acids inhibit secretion
from sea urchin eggs.
Source: Exp Cell Res
(EXPERIMENTAL CELL RESEARCH) 1983 Oct;
148 (1): 63-71 Journal
Code: EPB
Abstract: Massive secretion at
the egg surface follows fertilization of sea urchin eggs or
parthenogenetic activation by the
calcium ionophore A23187. The secretory
products are used to construct the
fertilization envelope around the egg. Arachidonic acid prevents the raising of the
fertilization envelope induced by
either sperm or A23187. We developed a
secretion assay based on the ability of A23187 to raise fertilization envelopes
from the surface of unfertilized eggs. Arachidonate delays the onset of this
reaction in a dose-dependent fashion. 5
microM arachidonate produces a two-fold
delay in the standard assay. In contrast, the propagation of secretion over the surface of the
egg is unaffected at all concentrations that
have been tested. Some closely related
fatty acids (e.g.
11,
14, 17 C20:3 and linoleate, 9, 12 C18:2) share
with arachidonate the ability to inhibit secretion, whereas others (e.g., 8,
11, 14 C20:3 and linolenate, 9, 12, 15
C18:3) do not. The results are not
easily reconciled with a cyclooxygenase- or a lipoxygenase-mediated action.
Despite the sensitivity of this
phenomenon to small changes in fatty
acid structure, it is suggested that the
fatty acids exert their effect by altering the structure or dynamics of
the membrane lipid bilayer.
Database: MEDLINE
·
Author(s):
Chambers EL ; Hinkley RE
Title: Non-propagated
cortical reactions induced by the
divalent ionophore A23187 in eggs of the sea urchin, Lytechinus
variegatus.
Source: Exp Cell Res
(EXPERIMENTAL CELL RESEARCH) 1979 Dec;
124 (2): 441-6 Journal
Code: EPB
Both of these experiments described in Foerder et al Proc Natl Acad Sci
75:3183-87.1978.
This assay will take advantage of the transphosphatidylation by the
enzyme, taking choline off PC and putting the ethanol on it as a head group.
The Ptd Eth can be detected on TLC. REFERENCES: D. Zoukhri, DA Dartt. 1995.
Cholinergic activation of PLD in lacrimal gland acini is independent of PKC and
calcium. Am. J Physiol 269 (3 pt1);c713-20.
Ella, KM, Dolan, JW, Meier, KE. 1995. Characterization of a regulated
form of PLD in the yeast S. Cerevisiae. Biochem. J. 307;799-805.
SPRECIFIC EXPERIMENT
Change in phospholipases at fertilization of sea urchin eggs.
NEED: SEA URCHIN EGGS AND SPERM,
If continuing from the previous fertilization lab start at the ***#4
below
SEA WATER, pH 8,
HOMOGENIZING
MEDIUM:(100 mM KCl, 5 mM MgCl2, 1 mM ATP, 10 mM benzamidine, 25 mM tris Cl pH
9.6), (Need 50 ml)
ETHANOL,
DETERGENT- octylglucoside
PLD reaction mix: (one
with and and one without 2% ethanol)
150 mM
NaCl
25 mM
HEPES pH 7
5 mM EDTA
1 MM EGTA
1 mM DTT
10 mM
substrate such as PC sonified in 200uM octylglucoside (make 10x, 2 ml) (Triton-x
inhibits enzyme, as does freezing)
ethanol or
not
Each group needs 4 ml. (20 ml total of solution.)
1. shed sea urchin
gametes into sea water by .6M KCl (in class had 15 ml eggs. treat with pH 5 3
min, adjust to pH 8 with tris, settle, then add new sea water so each group has
15 ml diluted eggs.) Take out 30 ml for unfert with and without Ca++.
2. fertilize the rest,
(add .1 ml diluted sperm per 10 ml) check to see if membrane lift away, place
on ice at a timed interval, like 5 min to stop development after
3. spin 15 ml in hand
centrifuge to pellet eggs, measure volume ( about 1.5 ml each),
****4.homogenize both unfert and fert batches using 10 ml
homog medium, keep cold. spin
20,000 rpm 10 min to get the membrane pellet.
5. place each pellet
(fert and unfert) in 2 ml PLD reaction
mixture and homogenize. KEEP COLD.
REACTION:prepare in a small eppendorf tube:
40 ul resuspended pellet with phospholipases
2ul
fluorescent PAF in detergent
2 ul
ethanol (add last)
add
additional things like 1 ul Ca++
This will make a fluorescent assay possible.
So have a control with only
Plipase, PAF, place in
tube,label well CONTROL. You should have one for the unfert, one for the fert.;
place a control with Plipase, PAF, ethanol in a different tube and mark
ETHANOL, one for fert, one for unfert, and an experimental with Ca++, Plipase,
PAF, ethanol in a different tube for both fert and unfert, mark Ca++. Now warm
them up!
7. Incub in white
incubator for 20-30 min at 35 degrees.
place 5 ul of sample on G60 silica gel plates (no Fluor marker)
Develop plate: chlor:meth:water/aCETIC ACID (45;45:10;1
PHOTO UNDER LONG WAVE UV WITH plate reader